Figure 4.

Depletion of endogenous PIP₂ produces a hyperpolarizing shift in activation of native and recombinant HCN3 channels. A, Exemplar I_h traces recorded from IGL neurons preincubated for 40–50 min in (Ai, control) or wortmannin (Aii, wort) before or after superfusion with 200 μm 8-Br cAMP. B, Group data showing steady-state activation curves. Lines are fit using a Boltzmann function to normalized tail currents, yielding the V_1/2 and slope factor (both in mV) of −89.2 ± 2.4 and 9.4 ± 1.1 for control (squares); −106.3 ± 3.5 and 8.2 ± 1.1 for wortmannin alone (circles); −105.6 ± 2.6 and 9.4 ± 0.9 for wortmannin + 8-Br cAMP (diamonds). Colored symbols used here represent the same conditions in subsequent panels for IGL neurons. There is a significant difference in V_1/2 values between control and wortmannin groups, p < 0.001, t test, n = 22. C, D, Effect of the drugs on the activation time constant (at −110 mV (C) and the current density at −130 mV (D). Almost identical effects are observed in IGL neurons from rats (n = 8) and HCN2^−/− mice (n = 10), and the two datasets were pooled. *p < 0.05, compared to control. E, HCN3 current traces recorded from intact oocytes following incubation in either control solution (which contains DMSO) (left) or wortmannin (right). F, Effects of wortmannin on steady-state activation curves; inset shows representative tail currents (left, wortmannin and right, control); red traces at −75 mV are for comparison. G, Negative shift in the V_1/2 of HCN3 channel activation following incubation with vehicle DMSO (control, n = 10) or wortmannin for 40 (n = 10) or 60 (n = 4) min. Vehicle controls were indistinguishable from the controls in the absence of DMSO (data not shown) in all experiments. The wortmannin-treated populations were significantly different from vehicle control and each other (*p < 0.05, vs control, one-way ANOVA with a Holm–Sidak post hoc test).